Journal: Nature protocols

Article Title: Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins

doi: 10.1038/s41596-018-0075-9

Figure Lengend Snippet: List of transfer plasmids.

Article Snippet: Proviral DNA sizes are noted in brackets. (D) Comparative size-exclusion chromatograms of secreted NET1 ΔNTR and NEO1 FN456 , showing improved secreted protein yields from lentiviral transduction (blue curves) as compared to transient transfection (red curves) of adherent HEK293T cells. table ft1 table-wrap mode="anchored" t5 caption a7 Plasmid no. pHR-CMV-TetO 2 Addgene Plasmid # Purpose 1 3C-Twin-Strep 113883 Protein (co-)expression, FACS 2 3C-Twin-Strep_IRES-EmGFP 113884 3 3C-Twin-Strep_IRES-mRuby2 113885 4 3C-Twin-Strep_IRES-mTurquoise2 113886 5 3C-Avi-His6 113887 Protein (co-)expression, FACS, in vivo biotinylation 6 3C-Avi-His6_IRES-EmGFP 113888 7 3C-Avi-His6_IRES-mRuby2 113889 8 3C-Avi-His6_IRES-mTurquoise2 113890 9 3C-mVenus-Twin-Strep 113891 Membrane protein expression and FSEC-TS screening, FACS 10 EmGFP 113892 Transduction & FACS compensation controls 11 mVenus 113893 12 mRuby2 113894 13 mTurquoise2 113895 14 HA-BirA 113896 HA-tagged cytosolic and ER-resident biotin ligase for in vivo biotinylation 15 HA-BirA-ER 113897 16 3C-Avi-His6_IRES-HA-BirA-ER 113898 Plasmid no. pHR-CMV Addgene Plasmid # Purpose 17 TetR-HA-NLS-P2A-BSD-Myc 113899 Generation of inducible cell lines Plasmid no. pHR-SFFV Addgene Plasmid # Purpose 18 3C-Twin-Strep 113900 Alternative for CMV-TetO 2 Plasmid no. pHR-CAG Addgene Plasmid # Purpose 19 3C-Twin-Strep 113901 Alternative for CMV-TetO 2 Open in a separate window caption a8 List of transfer plasmids.

Techniques: Plasmid Preparation, In Vivo, Membrane, Expressing, Transduction

Journal: Nature protocols

Article Title: Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins

doi: 10.1038/s41596-018-0075-9

Figure Lengend Snippet: (A-D) Multi-color FACS. (A) Non-concentrated lentiviral particles encoding β-NRX1LNS6_IRES-EmGFP (6.1 kb proviral DNA) and Cbln1FL_IRES-mRuby2 (6.0 kb proviral DNA) were used to co-infect HEK293T cells. (B) Fluorescent imaging indicated a mixture of non-, single- and double-transduced cells. The image was taken on a Leica SP8 SMD X confocal microscope. Scale bar: 50 μm. (C) Two-dimensional compensated fluorescence plot. (D) Western blot detection of His6-tagged β-NRX1LNS6(+SS4) and Cbln1FL secreted from co-infected cells after cell sorting (mouse anti-His6 primary antibody: 1:5,000 dilution, HRP-conjugated goat anti-mouse secondary antibody: 1:5,000 dilution). (E-F) In vivo biotinylation. (E) Non-concentrated lentiviral particles encoding either HA-BirA-ER (5.1 kb proviral DNA) or 3C-Avi-His6-tagged NL1ECTO (5.9 kb proviral DNA) were used to co-infect HEK293T cells. (F) Non-concentrated lentiviral particles encoding 3C-Avi-His6-tagged NL1ECTO and IRES_HA-BirA-ER (7.6 kb proviral DNA) were used to infect HEK293T cells. A D-biotin concentration of 100 μM was maintained in the DMEM/F-12/2%FBS medium. Anti-D-biotin western blot detection of in vivo biotinylated NL1ECTO (high sensitivity streptavidin-HRP conjugate; 1:4000 dilution). Anti-HA western blot detection of HA-tagged TetR (mouse anti-HA primary antibody: 1:5,000 dilution, HRP-conjugated goat anti-mouse secondary antibody: 1:5,000 dilution). (G-H) Generation of inducible cell lines. (G) Non-concentrated lentiviral particles encoding TetR-HA-NLS-P2A-BSD-Myc (5.1 kb proviral DNA) and mVenus (4.7 kb proviral DNA) were used to infect HEK293T and HEK293S GnTI– cells. (H) Flow cytometry analysis of the resulting HEK293Tlenti-TetR and HEK293S GnTI–lenti-TetR inducible cell lines, and comparison with the HEK293S GnTI– TetR cell line 34. MFI: mean fluorescence intensity.

Article Snippet: Proviral DNA sizes are noted in brackets. (D) Comparative size-exclusion chromatograms of secreted NET1 ΔNTR and NEO1 FN456 , showing improved secreted protein yields from lentiviral transduction (blue curves) as compared to transient transfection (red curves) of adherent HEK293T cells. table ft1 table-wrap mode="anchored" t5 caption a7 Plasmid no. pHR-CMV-TetO 2 Addgene Plasmid # Purpose 1 3C-Twin-Strep 113883 Protein (co-)expression, FACS 2 3C-Twin-Strep_IRES-EmGFP 113884 3 3C-Twin-Strep_IRES-mRuby2 113885 4 3C-Twin-Strep_IRES-mTurquoise2 113886 5 3C-Avi-His6 113887 Protein (co-)expression, FACS, in vivo biotinylation 6 3C-Avi-His6_IRES-EmGFP 113888 7 3C-Avi-His6_IRES-mRuby2 113889 8 3C-Avi-His6_IRES-mTurquoise2 113890 9 3C-mVenus-Twin-Strep 113891 Membrane protein expression and FSEC-TS screening, FACS 10 EmGFP 113892 Transduction & FACS compensation controls 11 mVenus 113893 12 mRuby2 113894 13 mTurquoise2 113895 14 HA-BirA 113896 HA-tagged cytosolic and ER-resident biotin ligase for in vivo biotinylation 15 HA-BirA-ER 113897 16 3C-Avi-His6_IRES-HA-BirA-ER 113898 Plasmid no. pHR-CMV Addgene Plasmid # Purpose 17 TetR-HA-NLS-P2A-BSD-Myc 113899 Generation of inducible cell lines Plasmid no. pHR-SFFV Addgene Plasmid # Purpose 18 3C-Twin-Strep 113900 Alternative for CMV-TetO 2 Plasmid no. pHR-CAG Addgene Plasmid # Purpose 19 3C-Twin-Strep 113901 Alternative for CMV-TetO 2 Open in a separate window caption a8 List of transfer plasmids.

Techniques: Imaging, Microscopy, Fluorescence, Western Blot, Infection, FACS, In Vivo, Concentration Assay, Flow Cytometry, Comparison